Rarely, humoral immunity generates broadly cross-reactive, neutralizing antibodies (NAb) to multiple isolates of HIV-1. Why are such antibodies so rare? Many NAb are autoreactive, leading to the hypothesis that effective humoral responses to HIV-1 are constrained by the tolerization of NAb B cells that also recognize self-antigens.

 

 Culture-derived (CD) murine B cells were expanded and matured with recombinant IL-7 and BAFF cytokines. Frequencies of self- and HIV-reactive B cells were determined by ELISpot and B-cell tetramer reagents decorated with MPER antigens. CD-RAG mice were made by reconstituting RAG-1-/- mice with CD B and T cells. CD-RAG and normal control mice were immunized with MPER antigen in adjuvant. Germinal center and antibody responses were determined ~2 weeks after 1-2 immunizations.

 

 We generated murine transitional B cells in vitro, outside the tolerizing environment of bone marrow (BM), and these CD cells contain substantial numbers (0.2-0.4%) of MPER B cells. Sorting MPER tetramer-binding CD B cells further enriches this population, allowing their molecular characterization. In vivo, the frequency of MPER transitional B cells in BM (0.43%±0.07%) was significantly higher than phenotypically identical cells in the spleen (0.15%±0.03%). Whereas immunization of C57BL/6 mice with MPER antigen did not elicit significant humoral responses, immunizations of CD-RAG mice produced significantly higher (3- to 4-fold) germinal center responses and MPER antibody. Control mice produced little MPER IgG antibody after immunization; in contrast, CD-RAG animals made ~10-fold higher quantities of MPER IgG.

 

 We show that HIV-1 MPER-reactive B cells are present at higher frequencies in vitro than in secondary lymphoid tissue. MPER-binding B-cells are lost during B-cell maturation, consistent with the tolerization of NAb by receptor editing or clonal deletion. This loss results in unresponsiveness to MPER antigens. The CD-RAG mouse is a novel animal model that allows investigation of “forbidden” humoral responses to HIV-1 MPER antigens.